1. Technical Field
Background interference can be a serious factor in diminishing the quantitative character of an immunoassay. In many situations, the background interference will vary from sample to sample and will be different in the analyte-containing samples than in the standards or calibrators employed for translating the observed signal into the concentration of the analyte. To enhance assay accuracy, it is desirable to diminish or remove the background interference contributed to the observed signal during the immunoassay.
This disclosure relates in particular to diminishing or removing background interference in immunoassays for the detection of amphetamine and methamphetamine, specifically interference caused by tyramine.
2. Background Art
Amphetamine and methamphetamine, the structural chemical formulas of which are presented below, are sympathomimetic phenethylamine derivatives having central nervous system stimulant activity. ##STR1##
They have been used for the treatment of obesity, narcolepsy, and hypotension. However, excessive or prolonged use may lead to tolerance and physical dependence. Because of their stimulant effects, these drugs are commonly sold illicitly. Physiological symptoms associated with high amounts of ingested amphetamine and methamphetamine include elevated blood pressure, dilated pupils, hyperthermia, convulsions, and acute amphetamine psychosis.
The biological fluid tested most frequently for the presence of amphetamine and methamphetamine is urine. Such substances have been detected by a number of techniques, including, for example, thin layer chromatography, gas chromatography, and high performance liquid chromatography. These methods require chemical extractions of the drugs from the urine sample, necessitating the use of labor intensive procedures requiring highly trained personnel and lengthy assay times.
For these reasons, immunoassays are a preferable alternative. Typically, in an immunoassay, for example, a competitive binding immunoassay, an analyte (a substance of biological interest to be determined quantitatively) competes with a labeled reagent, i.e. "an analyte analog," for a limited number of receptor binding sites on antibodies specific to the analyte and the analyte analog. The concentration of analyte in the sample determines the amount of analyte analog that binds to the antibody. The amount of analyte analog that binds to the antibody is inversely proportional to the concentration of analyte in the sample, because the analyte and the analyte analog each bind to the antibody in proportion to their respective concentrations.
An accurate and reliable immunoassay for amphetamine and d-methamphetamine requires that antibody "cross-reactivity" (i.e., recognition of compounds other than the desired analyte) be minimized. Although the d- and 1-enantiomers of methamphetamine are controlled substances in the United States, the 1-enantiomer has weaker stimulant activity and is contained in small amounts in some prescription and over-the-counter medications. It is therefore undesirable for an assay (in particular one employed to detect drug abuse) to respond to 1-methamphetamine alone in a sample. Therefore, the cross-reactivity for 1-methamphetamine should ideally be zero. Since 1-amphetamine is a major metabolite of 1-methamphetamine, it is also undesirable for the assay to respond to 1-amphetamine. Ideally, the assay will be specific for the d-enantiomers of both amphetamine and methamphetamine.
Other cross-reacting compounds, for example, derivatives of .beta.-hydroxyphenethylamine compounds, are known interferants in amphetamine and methamphetamine immunoassays. One such .beta.-hydroxyphenethylamine, phenylpropanolamine, is found frequently in decongestants and diet pills sold over the counter. U.S. Pat. No. 3,856,469 discloses removal of .beta.-hydroxyphenethylamine interference from a sample intended for amphetamine and/or methamphetamine analysis by treating the sample at a pH greater than 8.0 with an amount of aqueous periodate in the presence of ammonium hydroxide.
In addition, tyramine, which may be present naturally in a biological sample being analyzed for amphetamine and/or methamphetamine, may also be a strong interferant. Tyramine, p-hydroxyphenylethylamine, is a decarboxylation product of tyrosine, an amino acid found in most proteins. Tyramine interference from endogenous tyramine in an immunoassay can produce false positive results. The compositions and methods of the present invention significantly improve the selectivity of assays for d-amphetamine and d-methamphetamine (hereinafter alternatively termed amphetamine-class compounds or analytes) over tyramine in comparison with prior art methods.
For art relating to the detection of amphetamine and methamphetamine in biological samples, U.S. Pat. Nos. 3,996,344 and 4,016,146 (disclosing phenethylamine antigenic conjugates, their preparation, antibodies and use); 4,041,076 (disclosing immunoassay for pharmacologically active phenothylamines); 4,329,281 (disclosing hapten compositions for making immunogens usable to elicit antibodies selective to amphetamine and methamphetamine); 3,878,187 (disclosing polypeptide derivative amphetamine analogs for immunoassays); 5,101,015, 5,248,791, and 5,262,333 (disclosing reagents for use in fluorescence polarization immunoassay for amphetamine-class analyte detection) are exemplary. In addition, the scientific literature discloses a variety of amphetamine detection methodologies and reagents. See, for example, FEBS LETTERS 36:3 (1973) disclosing a radioimmunoassay procedure for measuring amphetamines in urine); Chem. Pharm. Bull. 25(4):840 (1977) (disclosing another radioimmunoassay for methamphetamine); Forensic Science International 27:49 (985) (disclosing a latex agglutination inhibition reaction test for screening urinary amphetamine); Clin. Chem. 32(9):1677 (1986) (disclosing a homogeneous fluoroimmunoassay for detecting amphetamines in urine; Jpn J. Legal Med. 37(4):417 (1983) (disclosing a solid phase micro-ELISA for methamphetamine); Chem. Pharm. Bull. 25(4):838 (1977) (disclosing preparation of a specific antibody to methamphetamine); and Clinical Toxicology, 18(1):91 (1981) (disclosing the analysis of amphetamine-related amines by the "EMIT".TM. procedure).
For art relating to reducing background interference in immunoassays generally, U.S. Pat. No. 4,668,620 (treatment of serum samples with peracids or persulfates to reduce interference caused by serum components other than analytes), U.S. Pat. Nos. 3,856,469 (use of periodate to reduce interference from .beta.-hydroxyphenethyl amines) and 5,262,333 (use of periodate to treat urine samples to reduce, inter alia, tyramine interference in immunoassays) are exemplary.